The long-term goal of this project is to investigate the structural basis of HIV protease drug resistance and to use that knowledge to devise strategies to overcome resistance. The novel hypothesis being tested is that HIV-1 protease drug resistance is caused by mutations that expand the active site of the protease and as a result the inhibitors bind with lower affinity to the variant forms of the enzyme. The short-term goal of this project is to study the structure and function of four multiple drug resistant HIV protease clinical isolates by using a combined structural, virological and biochemical approach. To accomplish this we will: Aim 1. Analyze the structures of inhibitor complexes of four multiple drug resistant "signature mutants" (807, 3761, 1385, 769) by using X-ray crystallographic and molecular modeling techniques. 30 modeled structures of protease variant-ligand complexes will be systematically compared to determine changes in inhibitor or substrate binding. One-third of the modeled structures will be verified by X-ray crystallography. Aim 2. Measure the virological (IC50) and enzymatic (Ki, Km, Vmax) indicators of drug resistance for a series of four multiple drug resistant HIV- 1 proteases (807, 3761, 1385, 769) and correlate the functional results with structural information derived from Aim 1. The structure-function data from the four multiple drug resistant clinical isolates will be used to examine drug resistance hypotheses including the novel HIV protease active site expansion hypothesis. If the active site expansion hypothesis is confirmed, it will suggest a logical approach to overcome drug resistance. Solving the structural problem of why some mutant HIV-1 proteases cannot be inhibited by the standard inhibitors and using this information to design new and effective compounds is of great importance to human welfare.